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Methods Mol Biol. 2008;416:279-93. doi: 10.1007/978-1-59745-321-9_18.

Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome.

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Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minaminohsawa, Hachioji, Tokyo, Japan.


Genetic information consists of protein- and RNA-coding genes that exist in a range of sizes and noncoding cis- and trans-acting sequence elements. The use of long chromosomal deletion mutations is a powerful method for identifying essential genetic information through experimental reduction of the genome to its minimal gene set. Taking advantage of recent technical advances, we constructed sequence-specific long deletion mutations of the Escherichia coli chromosome. In a recent report (1), we described a set of E. coli medium-scale deletions (MDs) and large-scale deletions (LDs). Several LD mutations were combined to generate an engineered strain lacking approximately 30% of the parental chromosome. We then constructed another set of deletion mutations, MDs and small-scale deletions (SDs), and identified additional essential genetic regions using complementation analysis. To delete the remaining essential chromosomal regions, we developed an Flp recombinase target (FRT)-based system of site-specific recombination to move chromosomal regions onto mini-F plasmids in vivo. In this report, we describe the details of the construction of several of these types of large chromosomal deletion mutants.

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